L-Arginine, N2-(2,3-dihydroxypropyl)-, monohydrochloride

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 February 2020 - August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with GLP and to the relevant OECD Test Guideline

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L and 0 mg/L (control group)
- Sampling method: Test samples were taken and analysed from the control and 100 mg/L test group from the bulk test preparation at 0 h and from the pooled replicates at 48 h. 10 mL of methanol was added to a 10 mL aliquot of sample. The samples were further diluted using methanol:test media (50:50 v/v) where necessary to keep concnetrations in the calibrated range.
- Sample storage conditions before analysis: The 0 h test samples were analysed on the day of sampling and stored refrigerated at approx. 4 ºC prior to analysis. The 48 h test samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the range-finder results (see 'Details on test conditions'), a limit test was conducted. A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 L to give a 100 mg/L test concentration.
- Eluate: N/A
- Differential loading: N/A
- Controls: Yes: A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test. The concurrent negative control group was maintained under identical conditions but not exposed to the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The stock solution prepared concentration was inverted several times to ensure adequate mixing and homogeneity. Preliminary solubility work showed that inversion was sufficient to allow the test item to fully dissolve.
- Other relevant information: N/A

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Not reported
- Age at study initiation (mean and range, SD): Under 24 h old
- Weight at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): Not reported
- Stage and instar at study initiation: First instar. Young daphnids produced overnight from gravid adults were used in the test.
- Method of breeding: First instar daphnids were derived from in-house laboratory cultures. Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4 medium in a temperature-controlled room, with water maintained at 18 to 22 °C. The lighting cycle was controlled to give a 16 h light and 8 h darkness cycle, with 20 min dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 h old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Source: In-house
- Age of parental stock (mean and range, SD): Not reported
- Feeding during test: No

ACCLIMATION
N/A

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Not reported

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

N/A

Hardness:

2.5 mmol/L (= 250 mg/L as CaCO3)

Test temperature:

19 - 20 °C

pH:

Control: 7.7 - 7.9
100 mg/L: 7.5 - 8.0

Dissolved oxygen:

Control: 9.1 mg/L
100 mg/L: 8.9 - 9.1 mg/L

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 100 mg/L; procedural recoveries 1 and 2: 105 %
Measured:
0 h: 93.4 mg/L; procedural recoveries 1 and 2: 101 %;
48 h: 102 mg/L
The concentrations for the procedural recovery are the actual fortified concentrations. The corresponding percentages of nominal concentration values relate to the percentage of fortified concentration recovered. Analysis of the test preparations at 0 and 48 h showed measured test concentrations to be near nominal, therefore the results are based on the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel
- Type (delete if not applicable): Covered, to reduce evaporation
- Material, size, headspace, fill volume: Size: 240 mL vessel, containing 100 mL test media
- Volume of solution: 100 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstitued water (Elendt M4 medium), see 'Any other information on materials and methods' for more information.
- Alkalinity: 0.9 mmol/L
- Water hardness: 2.5 mmol/L (= 250 mg/L as CaCO3)
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0, 24 and 48 h

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light and 8 h darkness was implemented for a period of 48 h, with 20 min dawn and dusk transition periods
- Light intensity: 797 - 840 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 h after the start of exposure. Daphnia were considered to be immobilised if they were unable to swim within 15 sec after gentle agitation.

VEHICLE CONTROL PERFORMED: N/A

RANGE-FINDING (R-F) STUDY
- Test concentrations: Nominal test concentrations: 0.1, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study:
The results of an initial range-finding test showed an inconsistent pattern in toxicity. In the initial test there was immobilization and trapping of the daphnia at the media surface seen, but this was not in a concentration dependent pattern. The results from this initial test was not used for reporting purposes. A further range-finding test was therefore performed.
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 L, to give the 100 mg/L test concentration. From this, a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
The stock solution and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity. The media was observed to be clear and colourless.

No sub-lethal effects of exposure were observed throughout the R-F test. Chemical analysis of the test preparations in the initial range finding test at 0 h showed measured test concentrations to range from 103 to 110% of nominal.
Based on the results of the range-finding test, a limit test was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no significant immobilisation was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

There was no immobilisation in 20 daphnids exposed to the test item (100 mg/L) for the duration of the test. No sublethal effects were observed throughout the test.
- Behavioural abnormalities: None observed
- Observations on body length and weight: Not reported
- Other biological observations: None observed
- Mortality of control: No mortality
- Other adverse effects control: None of the control daphnids showed immobilisation, or other signs of disease or stress.
- Immobilisation of control: No immobilisation
- Abnormal responses: None observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
-EC50: (48 h) 0.71 mg/L
- Other:
A positive control (Covance study number RQ16SK) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The positive control was conducted between 20 August 2019 and 22 August 2019.
Analysis of the immobilisation data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 h. All statistical analysis was carried out using the ToxRat Professional computer software package. The following results were obtained:

Time point EC50 95 % confidence limits NOEC LOEC
(h) (mg/L) (mg/L) (mg/L) (mg/L)
24 1.2 1.1 - 1.8 0.56 1.0
48 0.71 0.61 - 0.81 0.56 1.0

The NOEC is based upon equal to or less than 10% immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item (the results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24-Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L).

Reported statistics and error estimates:

Statistical analysis was not required, no effects were observed at or below the limit dose

The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Table 2. Cumulative Immobilization Data and Observations in the Definitive Test

Nominal concentration (mg/L)	24 h
	Cumulative immobilised daphnids (initial population: 5 per replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N
48 h
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

R = replicate

N = normal

Table 3. Water quality measurements

Nominal concentration (mg/L)		0 h			24 h	48 h
		pH	mg O2/L	Temperature ºC	Temperature ºC	pH	mg O2/L	Temperature ºC
Control	R1	7.7	9.1	19	20	7.9	9.1	20
100 mg/L	R1	7.5	9.1	20	20	8.0	8.9	20

R = replicate

 

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of 93 and 102 % of nominal respectively.

Validity criteria fulfilled:

yes

Remarks:

No more than 10 % of the control daphnids show immobilisation or other signs of disease or stress. Dissolved oxygen concentration at the end of the test was >= 3 mg/L in control and test vessels.

Conclusions:

Based on the conditions of the test, the test item EC50 was determined to be > 100 mg/L.

Executive summary:

A test was performed in accordance with OECD 202 (2004), in order to determine the acute aquatic toxicity of the test item to Daphnia magna.

Following preliminary range-finding tests, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item, in a limit test at a nominal concentration of 100 mg/L. The exposure duration was 48 h, conducted at a temperature of approx. 19 ºC to 20 ºC, under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 h. A negative control was run concurrently to the test. A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test. Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.

Chemical analysis of the test preparations at 0 and 48 h demonstrated measured test concentrations of 93 and 102 % of nominal respectively. Therefore, results were based on nomical concentrations. The results of the test were considered valid based on fulfillment of the following performance criteria:

• No more than 10 % of the control daphnids show immobilisation or other signs of disease or stress (e.g. discolouration or unusual behaviour such as trapping at the surface water).


• The dissolved oxygen concentration at the end of the test was >= 3 mg/L in the control and test vessels.

No sub-lethal effects and no mortality was observed during the test. The EC50 was determined to be > 100 mg/L following 48 h exposure. Under the conditions of this study, the test item does not cause acute toxicity to Daphnia. Based on Daphnia alone, the test item would not be classified under CLP Regulation (EC) 1272/2008.

Description of key information

OECD 202: EC50 > 100 mg/L (Ablitt, 2020)

Additional information

A test was performed in accordance with OECD 202 (2004) in order to determine the acute aquatic toxicity of the test item to Daphnia magna.

Following preliminary range-finding tests, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item, in a limit test at a nominal concentration of 100 mg/L for 48 h under static conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 h.

Chemical analysis of the test preparations at 0 and 48 h demonstrated measured test concentrations of 93 and 102 % of nominal respectively. Therefore, results were based on nominal concentrations. The results of the test were considered valid based on fulfillment the performance criteria as indicated in the OECD 202 guideline (2004):

• No more than 10 % of the control daphnids were immobilised and there were no other signs of disease or stress (e.g. discolouration or unusual behaviour such as trapping at the surface water).


• The dissolved oxygen concentration at the end of the test was >= 3 mg/L in the control and test vessels.

No sub-lethal effects and no mortality was observed during the test. The EC50 was determined to be > 100 mg/L following 48 h exposure. 

Under the conditions of this study, no effects were observed and therefore it was concluded that the test item does not cause acute toxicity to freshwater invertebrates. The test item would therefore not be classified under CLP Regulation (EC) 1272/2008 for acute aquatic toxicity, based on Daphnia magna alone.